It has become increasingly clear that second messengers play an important role in maintaining homeostasis in a diverse number of metabolic processes. Calcium is an important member of the group of second messengers, and regulation of calcium has become a focal point for investigating and controlling metabolic pathways and pathological conditions that can result from the aberrant regulation of these pathways. Regulation is achieved by opening or closing gated ion channels. This results in a change in the intracellular ion concentration in either of two ways: 1) changing the voltage across the plasma membrane or 2) allowing a major influx of ions, both generating an intracellular response. Calcium-regulated cell signaling pathways regulate cellular functions such as inflammation and smooth muscle contraction.
Inflammation is the body's reaction to injury. The inflammatory response involves three stages: first, an increase of blood flow to the injured area; second, an increase of capillary permeability caused by the retraction of endothelial cells lining vessel walls; and third, leucocyte migration to the site of injury. The third stage, known as chemotaxis, is a complex process that results in phagocytosis of invading agents by certain types of leucocytes such as the neutrophil. The neutrophil plays a key role in the body's response in inflammatory events such as infection. Once having arrived at the site of inflammation, the neutrophil is "activated" and releases a plethora of oxidative enzymes, known as a respiratory burst, that aid in destroying the invasive agent. An increase in intracellular calcium is thought to be involved in the initiation of the events that result in respiratory burst.
One group of compounds that have been shown to increase the influx of intracellular calcium in neutrophils is the "hepoxilins". Hepoxilins are products of an arachidonic acid pathway and have been implicated in the mediation of inflammation and smooth muscle contraction by modulation of second messenger calcium in response pathways. Hepoxilins are biologically active hydroxy epoxide derivatives of arachidonic acid formed through the 12-lipoxygenase pathway (Pace-Asciak et al., J. Biol. Chem. 258: 6835-6840, 1983; Pace-Asciak, Biochim. Biophys. Acta 793: 485-488, 1984; Pace-Asciak et al., Prostaglandins 25: 79-84, 1983). They are formed from 12-HPETE, an unstable hydroperoxide derivative of arachidonic acid (Pace-Asciak, J. Biol. Chem. 259: 8332-8337, 1984; Pace-Asciak et al., Adv. Prostal. Throm. Leuk. Res. 11: 133-134, 1983). Two hepoxilins have been isolated: hepoxilin A.sub.3 [ (S, R)-hydroxy-11(S),12(S)-epoxy-eicosa-5Z, 9E, 14Z-trienoic acid] and hepoxilin B.sub.3 [10(S, R)-hydroxy-11(R),12(S)-epoxy-eicosa-5Z, 8Z, 14Z-trienoic acid]. The term "hepoxilin" was coined in an attempt to combine aspects of structure with their first, though not necessarily their most important, demonstrated biological activity of insulin release (Pace-Asciak and Martin, Prostgl. Leukotriene and Med. 16: 173-180, 1984).
Hepoxilin A3 epimers are represented as: ##STR2##
Hepoxilin B3 epimers are represented as: ##STR3##
Hepoxilins are probably formed wherever 12-lipoxygenase is present because 12-HPETE is actively transformed into the hepoxilins by a variety of ferriheme proteins. Hence, ferriprotoporphyrin and such containing groups in proteins catalyze this transformation (Pace-Asciak et al., Biolog. Oxidation Systems, Eds C.C. Reddy et al., Academic Press, New York, 725-735, 1990). Hepoxilins are formed by platelets (Bryant and Bailey, Prostaglandins 17: 9-18, 1979; Jones et al., Prostaglandins 16: 583-590, 1978), lung (Pace-Asciak et al., Biochim. Biophys. Acta 712: 142-145, 1982), pancreatic islets (Pace-Asciak and Martin, ibid. 1984), brain (Pace-Asciak, ibid. 1988), aorta (Laneuville et al., Biochim. Biophys. Acta 1084: 60-68, 1991) and neutrophils (Dho et al., Biochem. J. 266: 63-68, 1990). Hepoxilin B.sub.3 has been isolated from marine red algae (Moghaddam et al., J. Biol. Chem. 265: 6126-6130, 1990) and hepoxilin A.sub.3 has been detected in the Aplysia brain (Piomelli et al., Proc. Natl. Acad. Sci. USA 86: 1721-1725, 1989). Hepoxilins are also formed by the rat pineal gland (Reynaud et al., unpublished observations).
Hepoxilins have been shown to possess a variety of biological actions related to their ability to affect ion fluxes in the cell. Hepoxilins raise intracellular calcium in human neutrophils (Dho et al., Biochem. J. 266: 63-68, 1990), increase the transport of calcium across membranes (Derewlany et al., Can. J. Physiol. Pharmacol. 62: 1466-1469, 1984), stimulate the release of insulin (Pace-Asciak and Martin, ibid. 1984), and regulate the volume of human platelets through an effect on potassium channels in the cell (Margalit et al. 1993 Proc. Natl. Acad. Sci. USA, in press). Biological actions of the hepoxilins demonstrated so far include the potentiation of aortic and tracheal vasoconstriction (Laneuville et al, Br. J. Pharmacol. 105: 297-304, 1992; 107: 808-812, 1992), potentiation of vascular permeability (Laneuville and Pace-Asciak, Prostaglandins, Leukotrienes, Lipoxins and PAF. Ed. J. M. Bailey, Plenum Press New York: 335-338, 1991), modulation of second messenger systems (Nigam et al., Biochem. Biophys. Res. Comm. 171: 944-948, 1990), regulation of cell volume (Margalit et al. Proc. Natl. Acad. Sci. USA, 1993, in press) and modulation of neurotransmission (Carlen et al., Brain Res. 497: 171-176, 1989; Piomelli et al., Proc. Natl. Acad. Sci USA 86: 1721-1725, 1989; and Pace-Asciak et al., Proc. Natl. Acad. Sci. USA 87: 3037-3041, 1990).
In view of the role of the hepoxilins in regulating physiological processes, a means for regulating hepoxilin action is needed. Hepoxilin antagonists find utility in reducing inflammation, asthma, hypertension, migraine and septic shock and in modulating other processes mediated by cellular calcium levels. Hepoxilin agonists find utility in diabetes.